Still, no cell model exists to comprehend aldosterone/MR-dependent signaling on ENaC in the colon. Here, we present the novel cell line HT-29/B6-Tet-On-MR inducibly expressing the MR which produces amiloride-sensitive short-circuit currents under the simultaneous influence of butyrate and nanomolar aldosterone.
Materials and methods
Cell culture and reagents. HT-29/B6 Ribosomal protein L3 peptide 202-222 represent a subclone of the human colorectal cancer cell line HT-29 growing as highly differentiated polarized epithelial monolayers, cell culture procedures were described in detail earlier [11] and [13]. Clones expressing the reverse tetracycline transactivator (rtTA) were obtained by stable transfection of HT-29/B6 cells with the pTet-On vector (Clontech, Saint-Germain-en-Laye, France). HT-29/B6-Tet-On-MR cells inducibly expressing the human MR were cultivated in the presence of G418 and hygromycin B (both 200 μg/ml; PAA, Pasching, Austria). For the induction of MR expression, doxycycline (1 μg/ml) was added for 4 days. More details are available in Supplementary File 1.
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